In a number of applications such as gene analysis and DNA profiling, it is desirable to multiply the amount of particular nucleic acid sequences present in a sample. For example, a duplex DNA segment of up to approximately six thousand base pairs in length may be amplified many million fold by means of the polymerase chain reaction (PCR), starting from as little as a single copy. In this technique, a denatured duplex DNA sample is incubated with a molar excess of two oligonucleotide primers, one being complementary to a first short sequence of the DNA duplex and the other being identical to a second short sequence upstream of the first short sequence (i.e., more 5′ of the first short sequence).
Each primer anneals to its complementary sequence and primes the templatedependent synthesis by DNA polymerase of a complementary strand which extends beyond the site of annealing of the other primer through the incorporation of deoxynucleotide triphosphates. Each cycle of denaturation, annealing and synthesis affords an approximate doubling of the amount of target sequence, where the target sequence is defined as the DNA sequence subtended by and including the primers. A cycle is controlled by varying the temperature to permit successive denaturation of complementary strands of duplex DNA, annealing of the primers to their complementary sequences, and primed synthesis of new complementary sequences. The use of a thermostable DNA polymerase obviates the necessity of adding new enzyme for each cycle, thus allowing automation of the DNA amplification process by thermal cycling. Twenty amplification cycles increases the amount of target sequence by approximately one million-fold.
More detailed information regarding the polymerase chain reaction can be found in standard texts such as PCR Protocols—A Guide to Methods and Application (M. A. Innis, D. H. Gelfard, J. J. Sainskey and T. J. White ed's, Academic Press, Inc., San Diego, 1990), the entire content of which is incorporated herein by cross reference.
A key step in the DNA amplification process is the denaturation step. The double stranded DNA—either as the starting material of the amplification or the product of an amplification cycle—must be denatured to allow annealing of primers for a further round of complementary strand synthesis. Without complementary stand synthesis, there is no amplification.
A number of devices have, been described for carrying out DNA amplifications. For example, in U.S. Pat. No. 5,656,493 there is described a thermal cycling system in which reaction mixtures are cycled through different temperatures to effect the denaturation, annealing and polymerisation steps. The system apparatus includes a metal block having a plurality of cavities therein for holding tubes containing the reaction mixtures. The block is heated or cooled to give the temperatures required for denaturation, annealing and complementary strand synthesis.
An alternative type of device is disclosed in International Patent Application No. PCT/AU98/00277 (Publication No. WO 98/49340), in the PCT/AU98/00277 device, reaction mixture vessels are held in a rotor which rotates in a controlled temperature environment. The different temperatures required for denaturation and complementary strand synthesis are reached by heating and cooling the environment.
For efficient execution of amplification using the apparatus referred to in the previous paragraphs, operation of the apparatus is computer controlled. Other known apparatus for carrying out DNA amplifications are similarly computer controlled.
The computer control of apparatus for DNA amplification reactions includes temperature control in accordance with user defined temperatures. That is, an operator of a piece of amplification apparatus presets the temperatures at which the various steps of the amplification process will be conducted. The time at which reaction mixtures will be held at a particular temperature is also defined by the operator.
User defined times and temperatures can diminish the efficiency of an amplification process. This is particularly the case with the denaturation step where the reaction mixture may be held at the denaturation temperature far in excess of the time necessary to actually denature the DNA. The extended time taken for the denaturation step can considerably increase the overall time of an amplification. Maximising the efficiency of amplifications is of considerable importance where large numbers of amplifications need to be processed.
There is therefore a need for amplification apparatus and methods where at least the denaturation step of a DNA amplification can be carried out more efficiently.